The overall objective of this proposal is an investigation of the oxidative metabolism of the human neutrophil with particular emphasis on the relationship of this metabolism to the bactericidal activity of the cell. We are especially interested in defining the metabolic basis of the respiratory burst and the factors which are responsible for the expression of this activity during phagocytosis. The following specific aims are directed toward this goal: 1. Characterization of a heavy granule fraction containing NAD(P)H oxidase activity using techniques of velocity and isopycnic density gradient centrifugation. We shall attempt to examine the various gradient fractions by electron microscopy as well as the use of marker enzymes. 2. Comparison of NADH and NADPH oxidase activities in cells from a number of patients with chronic granulomatous disease, acute bacterial infection, and polycythemia vera. 3. Attempt to delineate the mechanism of activation of the oxidase(s) by phagocytosis. We shall begin by examining the various gradient fractions for the presence of an enzyme inhibitor. 4. Continue efforts to purify the enzyme(s), using the gradient isolation procedure as an initial step. We shall next attempt to solubilize the activities obtained from the gradient by such techniques as detergent treatment, freeze-thawing, sonication, butanol extraction etc. If the enzyme(s) can be solubilized, further purification will rely upon conventional techniques of gel filtration, affinity chromatography etc. Only enzyme purification can unequivocally determine whether the cell possesses two separate oxidases or a single oxidase which can utilize either NADH or NADPH as substrate. BIBLIOGRAPHIC REFERENCES: DeChatelet, L.R., Shirley, P.S., and Johnston, R.B., Jr.: Effect of phorbol myristate acetate on the oxidative metabolism of human polymorphonuclear leukocytes. Blood 47:545 (1976). Wang, P., Shirley, P.S., DeChatelet, L.R., McCall, C.E., and Waite, B.M.: Purification of the plasma membrane from the BCG-induced rabbit alveolar macrophage. J. Reticuloendothel. Soc. 19:333 (1976).